Early life gut microbiota sustains liver-resident natural killer cells maturation via the butyrate-IL-18 axis

Liver-resident natural killer cells, a unique lymphocyte subset in liver, develop locally and play multifaceted immunological roles. However, the mechanisms for the maintenance of liver-resident natural killer cell homeostasis remain unclear. Here we show that early-life antibiotic treatment blunt functional maturation of liver-resident natural killer cells even at adulthood, which is dependent on the durative microbiota dysbiosis. Mechanistically, early-life antibiotic treatment significantly decreases butyrate level in liver, and subsequently led to defective liver-resident natural killer cell maturation in a cell-extrinsic manner. Specifically, loss of butyrate impairs IL-18 production in Kupffer cells and hepatocytes through acting on the receptor GPR109A. Disrupted IL-18/IL-18R signaling in turn suppresses the mitochondrial activity and the functional maturation of liver-resident natural killer cells. Strikingly, dietary supplementation of experimentally or clinically used Clostridium butyricum restores the impaired liver-resident natural killer cell maturation and function induced by early-life antibiotic treatment. Our findings collectively unmask a regulatory network of gut-liver axis, highlighting the importance of the early-life microbiota in the development of tissue-resident immune cells.

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All studies must disclose on these points even when the disclosure is negative. Reporting for specific materials, systems and methods Source data are provided with this paper. The 16S rRNA sequencing datasets were uploaded to the NCBI database with SRA, accession:PRJNA890468. RNA-seq data have been deposited in NCBI database under the accession number PRJNA884315. The raw data are provided as a Source Data file. The authors declare that all data supporting the findings of this study are available within the paper and its Supplementary Information files or from the corresponding author upon reasonable request.
Human normal liver tissue included in this study were obtained from 10 (5 females and 5 males) hepatic hemangioma patients in Qilu hospital, Shandong University and Shandong Provincial Hospital, and all patients provided informed written consent. Subjects elegible for this study were of all sexes (aged 26-65).
The normal liver tissues used in this study were from diagnosed hepatic hemangioma patients (aged 26-65).
Human liver tissue included in this study were obtained from 10 hepatic hemangioma patients in Qilu hospital, Shandong University and Shandong Provincial Hospital from May 2022 to September 2022. The authors do not see any potential bias in the generation or interpretation of the data reported in this study.
All human tissues used in this study were approved by the Ethics Committee of Shandong University Schoo of Basic Medical Sciences.
Sample sizes were chosen based on preliminary data demonstrating statistically significant differences for each specific assay No data was excluded.
For animal experiments, at least 5 mice were included for each group, and independent experimental repeats were performed two or three times to ensure reproducibility of the results. For in vitro experiment, replicates were not less than three in each group. All the replications showed the similar results.
For mouse experiment, mice were mated after 8 weeks of age and 4~5 male littermates from at least 2 dams were randomly assigned to different cages at post-weaning. Meanwhile, the offspring mice at same age and same gender from pregnant C57BL/6 dams without antibiotic treatment were maintained in parallel as controls in each experiment. For cell experiment in vitro, cells were randomly allocated to the experimental groups.
We had no specific methods to blind the investigators during the experiments, but all mice were treated equally at the same time

March 2021
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The mouse cell line Yac-1 cells was donated by Shandong Academy of Medical Sciences.
Yac-1 cells were used in vitro flow-based killing assay.
All cells were tested negative for mycoplasma contamination before use.
No commonly misidentified cell lines were used in the study.
C57BL/6 mice (6-8 weeks of age) were purchased from Beijing Vital River Laboratory Animal Technology. IL-18R! knockout mice were gifted by prof. Wei Wang from Sichuan University. CD45.1 mice were kindly provided by Dr. Xiaolong Liu (Center for Excellence in Molecular Cell Science, CAS). All mice were maintained under specific pathogen-free conditions, and experiments were carried out under the Shandong University Laboratory Animal Center's approval. All mice were maintained under specific pathogen-free conditions with a 12-h light, 12-h dark cycle and given free access to food and water. We analysed the function and maturation of LrNK cells both in weaning and 8-week-old control or early-Abx male mice. 8-week-old control or early-Abx male mice (18~22g body weight) were subjected to HCC models. IL-18R! knockout mice and CD45.1 mice (8week-old) were used in the experimental.
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There is no gender difference for the effect of early-life micorbiota on LrNK functional maturation. We have added this information in Material and Methods section The study did not involve field-collected samples.
Animal protocols were approved by the Shandong University Laboratory Animal Center. Animal Ethics Number: ECSBMSSDU2020-2-86.
For isolation of liver mononuclear cells (LMNCs), the mouse livers were washed and passed through a 200-gauge stainless steel mesh. The single cells were washed, red blood cells were lysed and then the cell suspension were centrifuged over 40% Percoll gradient medium. For bone marrow mononuclear cells, bone marrow was obtained by flushing femurs and tibias and then RBC lysed and washed with PBS. For cell surface staining, cell suspensions were incubated with the specific labeled antibodies for 30 min at 4°C. For intracellular staining, freshly isolated cells were stimulated with PMA (50ng/ml) (Sigma) and Ion (1#g/ml) (Biolegend) for 2h, or IL-12 (20ng/ml) (Proteintech) and IL-15 (50ng/ml) (Proteintech) for 16h, then cultured with Brefeldin A (BFA) (Biolegend) at a final concentration of 10 #g/mL for 4h. After surface staining, cells were fixed with intracellular fixation buffer for 20min, then permeabilized with permeabilization buffer for 10min. Intracellular staining was performed with antibodies diluted into permeabilization buffer. For CD107a staining, cells were incubated with CD107a antibody for 4h. For purification of LrNK cells, Kupffer cells, IL-18R!-LrNK and IL-18R!+LrNK cells, LMNCs isolated as described above were stained with surface marker antibodies and subjected to Moflo Astrios EQ.